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Contents lists available at ScienceDirectJournal of Plant Physiology
journal homepage: www.elsevier.comCaspase-3-like activity and proteasome degradation in grapevine suspension cell
cultures undergoing silver-induced programmed cell death
Antonio Filippi, Marco Zancani, Elisa Petrussa, Enrico Braidot
⁎A R T I C L E I N F O
Keywords: Caspase-like proteins Programmed cell death Proteasome
Reactive oxygen species Silver metal
Vitis vinifera
A B S T R A C T
Toxic metal contamination is one of the major environmental concerns of the recent decade, due to the large application of metals in industrial, healthcare and commercial products, even in the form of nanostructures and nanomaterials. Nevertheless, the effects of silver (Ag+) on plants have not yet thoroughly elucidated. Therefore,
suspension cell cultures of grapevine were used as a model for investigating silver toxicity. To do this, oxidative stress and programmed cell death (PCD), evaluated as reactive oxygen species production, caspase-3-like activity and ubiquitin-proteasome system, were investigated.
As a result, the highest concentration (10 µM) of Ag+caused a rapid (within 24 h) induction of PCD
(ap-prox. 80%), accompanied by generation of reactive oxygen species and activation of caspase-3-like activity. In the presence of specific inhibitor of this enzyme, a partial recovery of cell viability and a strong inhibition of caspase-3-like activity was observed. In addition, silver-induced PCD was accompanied either by increase of poly-ubiquitin conjugated proteins and degradation of subunit PBA1 of the proteasome 20S core, similarly to what found for metal-induced neurotoxicity in animals.
The present study shows that silver could induce PCD in grapevine suspension cell cultures, mediated by caspase-3-like activity and oxidative stress. These effects were associated to accumulation of poly-ubiquitin con-jugated proteins, suggesting the impairment of ubiquitin-proteasome complex, confirmed by the decrease of the PBA1 subunit. These findings indicate that animal and plant cells could share a common pathway in response to toxic metal, which involves PCD and disassembling of proteasome complex.
1. Introduction
Programmed cell death (PCD) is an integral part of multicellular organism’s life cycle. This process is induced by various stimuli (de-velopmental and environmental cues), involving the expression of dif-ferent genes in specific sub-cellular compartments (endoplasmic reticu-lum, plasma membrane, vacuole, mitochondrion and chloroplast) (Cai et al., 2014; Li et al., 2010; Üstün et al., 2016). Plants exploit PCD during different developmental processes, such as senescence, embryo-genesis, development of vascular tissues and sex determination in
uni-sexual plants (Van Aken and Van
Breusegem, 2015; Xu et al., 2005). PCD represents also a key mech-anism in plant-pathogen interaction, because it can be locally elicited where the tissue is invaded, reducing the growth of the pathogen (Jiao et al., 2014; Kim et al., 2003).
It is well known that multiple forms of PCD exist in both plants and animals, sharing a number of similar hallmarks, such as DNA fragmen-tation and cell shrinkage (Kim et al., 2003; Vartapetian et al., 2011; Zancani et al., 2015).
Many observations suggest that oxidative stress and generation of reactive oxygen species (ROS) may be an early event of plant re-sponse to several biotic and abiotic stresses, leading to PCD (Huang et al., 2014). The cellular sites of the most important meta
Abbreviations: U, arbitrary units; Ac-DEVD-pNA, N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide; DEVD-CHO, Asp-Glu-Val-Asp-aldehyde; DTE, 1,4-dithioerythritol; FDA, fluorescein diacetate; GSC, grapevine suspension cell culture; H2DCFDA, 2′, 7′-dichlorodihydrofluorescein diacetate; HM, heavy metal; pUCP, poly-ubiquitin conjugated proteins; PCD, programmed cell death;
PBA1, proteasome beta A1 subunit; ROS, reactive oxygen species; 20SP, 20S proteasome; Ub, ubiquitin; UPS, ubiquitin-proteasome system.
⁎ Corresponding author at: Dept. of Agricultural, Food, Environmental and Animal Sciences (Di4A), University of Udine, Via delle Scienze 91, 33100 Udine, Italy.
Email address: [email protected] (E. Braidot)
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bolic processes, such as chloroplasts and mitochondria, are also thema-jor ROS producers and may generate intermediate signals involved in PCD (Borutaite and Brown, 2001; Li and Xing, 2011; Van Breusegem and Dat, 2006).
A general feature of PCD is the triggering of key enzymes, such as caspases, a family of cysteine-proteases, occurring as proenzymes in most animal cells (Fuentes-Prior and Salvesen, 2004; Kumar, 2007) and known as caspase-like in plants, due to their different sequence respect to mammalian counterpart (Cai et al., 2014; Chichkova et al., 2012; Xu and Zhang, 2009). The action of the active caspases on their sub-strates causes morphological changes leading to cell death (Kim et al., 2003). Considering that organisms belonging to different kingdoms pre-sent strong similarities in these processes, it was expected that also some regulators and executioners of animal apoptosis could be still conserved in plant PCD manifestation and signalling (Fomicheva et al., 2012). However, no animal caspase orthologues have been found in plant genomes and the plant proteases closest to animal caspases, the meta-caspases, are unable to cleave synthetic caspase substrates (Salvesen et al., 2015). The plant caspase-like activities, therefore, originate from proteases structurally unrelated to animal caspases, although the identi-fication of plant proteins responsible for proteolytic caspase-like activi-ties is still in progress (Cai et al., 2014; Schaller, 2004).
Two major pathways for caspase activation have been so far defined in metazoans: the extrinsic death receptor pathway and the intrinsic or mitochondrial pathway (Kumar, 2007). The intrinsic pathway starts with the induction of assembly of the caspase-9 activating complex, named proteasome. Upon proteasome activation, caspase-9 can propa-gate a cascade of further caspase activation events by direct processing of effector caspases. Proteasome-involving machinery is one of the main protein degradation systems of eukaryotic cells that not only removes misfolded and defective proteins, but also controls various cellular path-ways through the selective removal of short-lived regulatory proteins (Üstün et al., 2016).
In plants, proteasome (26S proteasome complex) is composed by 31 polypeptides, organized in two sub-complexes. The first is the ATP‐ and ubiquitin (Ub)‐independent catalytic core particle, or 20S proteasome (20SP), where proteins are degraded. The second complex is composed of two regulatory particles, or 19S proteasomes, which cap the 20SP and confer ATP dependence and Ub recognition (Sadanandom et al., 2012). Recent studies evidenced a possible involvement of 20SP in plant velopmental PCD, as shown in poplar where xylem development is de-pendent on a caspase-3-like activity associated to this proteasome com-ponent (Han et al., 2012). This finding supports the view that some as-pects of the molecular machinery of PCD seem to be conserved between plants and animals (Kim et al., 2003). On the other hand, Hatsugai et al. (2009) demonstrated that caspase-3-like proteolytic activity in Arabidopsis could be attributable to 20S proteasome beta A1 subunit (PBA1) also in bacteria-induced PCD. These authors showed that PBA1 mediated the fusion of plasma membrane to tonoplast, leading to vacuo-lar autophagy. In addition, proteasome inhibition seems to be involved in cell death pathway in Arabidopsis, since proteasome disruption leads to a PCD form that is characterized by ROS production and caspase‐like activation (Kurepa and Smalle, 2008). Furthermore, UPS is required for defence priming and the proper establishment of systemic acquired re-sistance, by preventing futile defence activation (Matsushita et al., 2013; Spoel et al., 2009). In addition, bacterial effectors are able to inter-fere with plant proteasome, causing to inhibition of its activity and thus leading to the pathogenic status (Üstün et al., 2016).
On the other hand, the role of proteasome in inducing the degrada-tion of misfolded proteins is believed to be the key point in cellular pro-teomic stability, the impairment of which could increase the engulfment of subcellular compartments and thus leading to PCD (Li et al., 2010).
Among the environmental stresses that plants could face, toxic heavy metals (HMs) have been demonstrated to be strong triggers of PCD (Hasan et al., 2017). Despite the huge production and dispersion of metal-based salts and nanomaterials into the environment, the role of polluting metals from anthropic origin, in relation to their phytotox-icity, has not yet been fully investigated. These metals include ele-ments like silver, mercury, lead, arsenic, cadmium, titanium and zinc (Vishwakarma et al., 2017; Yu et al., 2011). In particular, silver is widely utilized as metal nanoparticles, either occurring in a large array of healthcare antimicrobial products and outdoor facades formulated products (Kaegi et al., 2010; Mueller and Nowack, 2008).
Metal ions at high concentrations, exceeding the limit in plant tis-sues of 5 ppm As3+, 2 ppm Cd2+, 18 ppm Cr6+, 46 ppm Cu2+, 3 ppm
Pb2+, 200–1000 ppm Al3+, 0.1–10 ppm Co2+, 100 ppm Mn2+, 4 ppm
Ni2+and 160–300 ppm Zn2+, (Hajar et al., 2014; Yadav, 2010), could
affect cellular protein homeostasis, modifying the biological functions of proteins by altering their native conformation, stimulating aggrega-tion of nascent proteins, leading to endoplasmic reticulum modificaaggrega-tions and decrease of cell viability, probably by interference with the UPS (Dametto et al., 2015; Hasan et al., 2017).
It is generally accepted that a marker of plant response to HMs stress is also represented by the modulation of Ub gene expression (Hasan et al., 2017). Moreover, recent studies showed the involvement of caspase-like activities, DNA fragmentation and mitochondrial dys-functions during PCD events caused by different HMs (Dametto et al., 2015; Iakimova and Woltering, 2017; Karmous et al., 2014; Petrov et al., 2015).
Interestingly, in a study on the effects of environmental HM exposure on animal neurodegenerative disease development (Yu et al., 2011), the activation of protein turnover alteration was reported. Conservation of a common response to HM toxicity shared between animals and plants could be very suggestive.
The present work aimed at evaluating the PCD manifestation in-duced by silver in a simplified plant cell model. We also investigated the degradation of proteasome complex subunit in association to PCD when grapevine cell cultures were exposed to silver ion.
2. Material and methods
2.1. Plant material and culture conditions
Dark-grown grapevine (Vitis vinifera L., cv. Limberger) callus cultures were maintained as described in Bertolini et al. (2016).
For the assay experiments, 4 g (FW) of 24-d-old cultures was trans-ferred into 100 mL of modified liquid Murashige-Skoog medium into 250 m L-flask. For silver treatment, different concentrations of AgNO3
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2.2. Determination of PCD
At different sampling times, 0.5 mL aliquots of GSC (approx. 20 mg FW) were incubated with 8 µg/mL fluorescein diacetate (FDA) for 5 min in the dark, and observed under LEICA Fluovert microscope (Bertolini et al., 2016). For each replicate, at least 100 cells were evaluated, and only FDA-stained green fluorescent cells were considered alive. The per-centage of PCD was calculated as the ratio of dead cells undergoing cy-toplasmic shrinkage, compared to the total cell number, as reported by Hogg et al. (2011).
2.3. Determination of in vitro caspase-3-like activity
At different sampling times, GSC were filtered by nylon gauze (100 µm), immediately ground to powder with liquid nitrogen and stored at −80 °C. The powder (approx. 3 g FW) was re-suspended in 3 mL of 20 mM MOPS–KOH (pH 7.5), 1 mM EDTA, 5 mM 1,4-dithio-erythritol (DTE) and 0.1% (w/v) CHAPS, and incubated on ice for 30 min. After centrifugation at 14,000 g for 30 min (Mikro 120 Het-tich centrifuge), cytosolic proteins were concentrated by 10,000 MWCO concentrators (VIVASPIN 6, Sartorius, Göttingen, Germany) at 8000 g for 30 min. Soluble proteins (approx. 10 µg) were used for the colori-metric assay of caspase-3-like activity in 100 mM sodium acetate (pH 4.5), 0.1% (w/v) CHAPS, with 25 µM N-Acetyl-Asp-Glu-Val-Asp p-ni-troanilide (Ac-DEVD-pNA, from a 10 mM stock solution in dimethyl sul-foxide) as substrate. The absorbance at 405 nm was measured after 5 h of incubation at 32 °C, using a 96-well microplate in a final reaction volume of 200 µL by Multilabel Plate reader (VICTOR3, Perkin Elmer,
Waltham, USA). 2.4. ROS determination
ROS production was measured according to Bertolini et al. (2016), using 2′, 7′-dichlorodihydrofluorescein diacetate (H2DCFDA) as a probe.
Powder of GSC was extracted in 10 mM Tris-HCl pH 7.3 with a ratio of 1:1 (w/v), centrifuged at 14,000 g for 10 min and the supernatant, cor-responding to 14 µg protein, was incubated in 96-well microplate with 5 µM H2DCFDA in a final reaction volume of 200 µL. The detection was
performed after 2.5 h incubation in the dark, using a Multilabel Plate Reader (excitation and emission wavelength set at 485 nm and 535 nm, respectively). Fluorescence A.U. (Arbitrary Units) were normalized by mg protein.
2.5. Immuno-detection of PBA1 and of poly-ubiquitin conjugated protein (pUCP)
GSC were obtained as above described for caspase-3-like activity assay. Soluble proteins (15 µg) were separated under reducing condi-tions (10 mM 1,4-dithioerythritol, DTE) by 12% (w/v) and 7.5% (w/v) SDS-PAGE for detection of PBA1 and Ub, respectively, and electro-blot-ted onto nitrocellulose membrane. The blots were incubaelectro-blot-ted at room temperature overnight with either a polyclonal antibody raised against PBA1 (anti-PBA1, 1/1000 dilution; Abcam, Italy), or with a polyclonal antibody raised against Ub (anti-UBQ11, 1/:10,000 dilution; AGRISERA, Sweden). The cross-reaction was detected by nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate staining, after incubation with alkaline phosphatase-conjugated anti-rabbit IgG antibody (1/2500 di-lution; Sigma, St. Louis, MO, USA). Computer-assisted densitometric analysis of immunoblots was performed using Quantity One software (Bio-Rad, Hercules, USA).
2.6. Determination of antioxidant metabolites
For the detection of carotenoids, GSC were sampled at 0 and 24 h, frozen in liquid nitrogen and ground with mortar and pestle to ob-tain powder. Fifty mg of powder was resuspended and homogenized in 0.5 mL of 90% (v/v) acetone and 0.01 N NH4OH. Then 0.5 mL of 80%
(v/v) acetone was added. The insoluble components were separated by centrifugation at 16,000 g (Sorvall rotor HB-4) for 10 min. The super-natant was collected and used for the spectrophotometric analysis at 480 nm by a diode spectrophotometer 8453 (Agilent, Santa Clara, CA, USA). The carotenoid content was determined according to the method proposed by Wellburn (1994).
Flavonoid measurement was performed following the method de-scribed by Filippi et al. (2015). Briefly, GSC powder obtained as above described was resuspended in 400 µL of cold methanol. DPBA (2-aminoethoxydiphenyl borate, 0.05%, w/v, in ethanol) was added to samples with 1:2 ratio in a final reaction volume of 200 µL. The detec-tion of fluorescence was performed after 2.5 h of incubadetec-tion in the dark, using a Multilabel Plate reader (excitation and emission wavelength set at 465 nm and 535 nm, respectively). The amount of flavonoids detected in GSC powder was expressed as equivalent of quercetin.
2.7. Protein quantification
The protein concentration assay was carried out according to the method of Bradford (1976), using bovine serum albumin as standard. 2.8. Statistical data analysis
Data were analysed as means of at least three independent replicates. The effects of different AgNO3concentrations were analysed by using
one-way ANOVA test, whereas the effects of incubation times (0, 6 or 24 h), treatment (± 10 µM AgNO3or ± 10 µM DEVD-CHO) and their
interactions were compared by two-way ANOVA test. Treatment means were compared by Least Significant Difference (LSD) analysis, accord-ing to Fisher’s statistical test, and different letters, assigned to means, indicate a statistical difference at P ≤ 0.05. All statistical analyses were performed using the Statistica software ver. 10 (Statsoft Inc., Tulsa, OK, USA).
3. Results
3.1. Characterization of grapevine callus cultures and GSC
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3.2. Effect of AgNO3on GSC cell death
After the transfer into liquid medium, GSC were used for investigat-ing the effects of different AgNO3concentrations and times of treatment
on PCD induction. The effects of silver on GSC were evaluated after in-cubation with 0.1, 1, or 10 µM AgNO3for 3 d. Living cells were
eval-uated by green fluorescence after FDA staining, while non-fluorescent cells, under visible light, showed either cytoplasm shrinkage (PCD) or swelling (necrosis), respectively (Fig. 1). Since the highest concentration of AgNO3(10 µM) induced death in almost all cells after 24 h, we
de-cided to follow PCD time-course only within this period of incubation. Fig. 2 shows that a significant level of PCD occurred after 24 h of in-cubation in 10 µM AgNO3-treated GSC, if compared to the level of
con-trol at 24 h, increasing PCD up to 80%. The overall effect of Ag+
con-centration on PCD was found to be highly significant (p < 0.001) (Ad-ditional file 2, Table 1). In both control and 0.1 µM AgNO3-treated
sam-ples, a slight increase in PCD level was also detectable at 24 h, although the difference with that observed at time 0 was not statistically signif-icant. Most importantly, the characterization of time-course of PCD in GSC treated with 10 µM AgNO3showed 40% of PCD already after 6 h of
incubation (Fig. 2C).
Consistently, the percentage of necrotic cells did not change in treated-cells after 24 h, even at the highest Ag+ concentration,
since necrosis remained near to basal level of approx. 20% (Fig. 2B and D). The range of concentration of Ag+was thus sufficient to induce
a program of cell dismantling, without provoking a massive leakage from cell membranes and swelling. As shown in Fig. 2D, the percentage of necrotic cells evaluated in control samples changed over time, being significantly decreased after 24 h; similarly, ANOVA test confirmed that variance of time x concentration was significant (p < 0.05; Additional file 2, Table 2).
3.3. Effect of AgNO3on ROS production
The manifestation of PCD in plants is usually associated to oxidative burst, due to an increase of intracellular ROS generation (Petrov et al., 2015). In this regard, oxidative stress induced by Ag+on GSC was
eval-uated as increase of fluorescence due to H2DCFDA complexation with
ROS, after cleavage by cytosolic esterases. ROS production in GSC ex-tracts exhibited a significant increase only in response to 10 µM Ag+
(Fig. 3A), whereas lower concentrations of Ag+(0.1 and 1 µM) showed
a slight, not significant, decrease when compared to untreated sample at 24 h. Particularly, the effects of low Ag+concentrations on ROS
produc-tion were overlapped by those due to complexaproduc-tion with ROS induced in liquid culture assessment. Treatment with 10 µM AgNO3did not change
ROS production (Fig. 3B and Additional file 2, Table 1) in GSC after 6 h of incubation, even if concomitantly the level of PCD was significantly increased (Fig. 2B).
Fig. 1. Microscopic observation of PCD and necrosis in GSC incubated with AgNO3. GSC were grown for 24 h in the presence of 10 µM AgNO3. After FDA staining, cells were observed
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Fig. 2. Effect of AgNO3on PCD and necrosis in GSC. A) Evaluation of PCD in GSC grown for 0 or 24 h, in the presence of different concentrations of AgNO3, respectively. B) Evaluation
of necrosis in GSC grown for 0 or 24 h, in the presence of different concentrations of AgNO3, respectively. C) PCD in GSC grown for 0, 6, or 24 h in the presence or in the absence of 10
µM AgNO3. D) Necrosis in GSC grown for 0, 6, or 24 h in the absence or in the presence of 10 µM AgNO3. Values are means ± S.D. of at least three replicates. Means were evaluated
by one-way (effect of AgNO3concentration) or two-way (effect of AgNO3concentration and incubation time) ANOVA and compared by LSD (Least Significant Difference), according to
Fisher’s statistical test. Different letters indicate a significant difference (p ≤ 0.05).
3.4. DEVD-dependent caspase activity induced by AgNO3
Since 10 µM AgNO3induced PCD in GSC, we investigated if the
process could involve the expression and activation of caspase-like pro-teases. Consistently, it has been demonstrated that caspase-3-like en-zymes are directly involved in some developmental plant PCD processes (Ge et al., 2016; Han et al., 2012). Therefore, DEVD-dependent cas-pase-like activity was evaluated in GSC treated for 24 h with different concentrations of AgNO3(Fig. 4). A basal level of caspase-3-like
activ-ity was already detectable in untreated GSC at both 0 and 24 h, due to the presence in control samples of about 20% PCD level. However, the caspase-3-like activity strongly increased after 24 h in GSC treated with 10 µM AgNO3(Fig. 4A), when compared with the lower concentrations,
showing a significant difference (p < 0.001, additional file 2, Table 1). Furthermore, when the experiment was performed at 6 h (Fig. 4B), this activity was significantly affected by both incubation time and silver concentration, as well as their interaction (Additional file 2, Table 2).
The involvement of caspase-3-like enzymes in PCD induced by silver was further investigated by adding the caspase inhibitor (DEVD-CHO) to GSC. In samples treated for 24 h with 10 µM AgNO3in the
pres-ence of the caspase inhibitor, PCD was significantly lowered from 80 to 60% (Fig. 4C and Additional file 2, Table 2, with p < 0.001). It is noteworthy that DEVD-CHO did not significantly alter the
per-centage of necrotic cells in both treated
and untreated samples at 24 h (Additional file 2, Table 1). In addi-tion, DEVD-CHO strongly inhibited in vitro caspase-3-like activity in Ag+-treated GSC after 24 h (Fig. 4D), which is in agreement with the
ob-served decrease in PCD. A slight effect of DEVD-CHO was also obob-served in the case of the basal caspase-like activity present in untreated cells (Fig. 4D).
3.5. Accumulation of pUCP and degradation of PBA1 induced by AgNO3
Since poly-ubiquitinated by-products are typically accumulated when proteasome system is disrupted or not functional, their detec-tion represents a suitable diagnostic tool for cell metabolism impair-ment (Speranza et al., 2001). This analysis was, hence, performed in ex-tracts obtained from GSC to verify if silver could also affect the func-tion and the structure of proteasome system. The detecfunc-tion of pUCP, evaluated by immunoblotting, was used as a marker of proteasome sys-tem alteration (Fig. 5). In agreement with the above results, GSC incu-bated for 24 h with 1 and 10 µM AgNO3showed a significant increase
in pUCP accumulation (Figs. 5A and 6A), as shown by a p value < 0.001 in ANOVA for both concentration and time factors, as well as for their interaction (p < 0.01) (Additional file 2, Table 2). These find-ings suggest that the proteasome-dependent proteolysis cascade was im-paired during PCD induced by Ag+. Noteworthy, GSC treated with 1
µM AgNO3for 24 h showed a significant increase of pUCP
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Fig. 3. Effect of AgNO3on ROS production in GSC extracts. A) ROS production in GSCgrown for 0 or 24 h, in the presence of different concentrations of AgNO3, respectively. 3B)
ROS formation in GSC grown for 0, 6 or 24 h in the absence or presence of 10 µM AgNO3.
Values are means ± S.D. of at least three replicates. Statistical analysis was the same as in Fig. 2.
ubiquitination significantly declined in untreated GSC from 6 to 24 h, whereas it appeared significantly higher in Ag+-treated samples (Figs.
5C and 6C).
The detection of PBA1 is considered a reliable marker of integrity of the proteasome, the complex responsible for recognition and cleavage of ubiquitinated proteins, since such a subunit is involved in the correct assembly of the 20SP complex in mammals (Hirano et al., 2008). There-fore, we used an anti-PBA1 antibody to quantify this component dur-ing Ag+-induced PCD in GSC (Figs. 5 and 6, Additional file 2, Tables 1
and 2). The results demonstrate that only when GSC were incubated for 24 h with 10 µM AgNO3there was a significant decrease in PBA1 (Figs.
5B and 6B), although the degradation was already initiated when GSC were incubated with 1 µM AgNO3(p < 0.001, additional file 2, Table
2). PBA1 level did not change in GSC untreated or treated with 10 µM AgNO3, analysed at 6 h (Figs. 5D and 6D).
4. Discussion
Recent advance about the use of metal nanomaterial in agro/med-ical and technologagro/med-ical fields opens new questions regarding release of appreciable amounts of different metal ion species (e.g. Au, Ag or Ti) in the environment (Vishwakarma et al., 2017; Yu et al.,
2011). Metal nanostructures are expected in the next future to be used in the agronomic field directly as plant nutrients or as shuttles for ac-curate delivery of nutrient or phytochemical to plant, as well as envi-ronmental remediation. For these reasons, it is conceivable that metals, usually present at low concentrations in nature, might raise at high level in soils in the next future. It is well-known that metal pollution impairs plant metabolism (Hasan et al., 2017), although phytotoxicity is strictly dependent on detoxification strategies, as well as on metal concentra-tion, plant age and environmental source (Nagajyoti et al., 2010).
In this regard, our study aimed to determine the phytotoxicity of silver (in form of silver nitrate, AgNO3) in Vitis vinifera, as one of the
most important crop plant. Ag+is not generally considered as pollutant
in natural conditions, but it is largely used as nanomaterial in indus-trial processes (Kaegi et al., 2010) and may represent in the next fu-ture a significant part of nanomaterial waste. In our experimental plan, Ag+ toxicity on Vitis vinifera cell cultures was evaluated at
micromo-lar concentrations of nitrate salt (0.1, 1, 10 µM, corresponding to 0.01, 0.1, 1 ppm, respectively), after incubation up to 24 h. The application of AgNO3to GSC was used utilized here as an in vitro bioassay test to
investigate the possible direct interactions of Ag+with homogeneous,
reproducible and undifferentiated cell system. Although several works report that Ag+and Ag nanoparticles inhibit root growth and alter their
morphology, as well as they modify seed germination, these studies did not explore the possible effects of such metal on cell death (Pallavi et al., 2016; Yin et al., 2012). On the other hand, although PCD induced by HMs in plants has been already reported (Hasan et al., 2017), few works have dealt with Ag+induction of cell death (Vishwakarma et al., 2017),
e.g. in mustard plant, where both ionic and nanoparticle forms of Ag+
cause DNA damage and PCD. In the present work, we verified that mi-cromolar concentrations of AgNO3(10 µM) induced approximately 80%
of PCD after 24 h of treatment and we showed that PCD induction was positively correlated to Ag+concentrations and incubation time (Figs. 1
and 2). We suggest that the higher sensitivity to Ag+exhibited by GSC
relies on the differences between our simplified system and the complex situation described in plants by Vishwakarma et al. (2017).
In plants, PCD manifestation is accompanied by formation of ROS, whether or not the latter represent the cause or the effect of PCD. ROS increase is considered as a signal linked to the induction of mito-chondrial membrane permeability alteration, which not only decreases oxidative phosphorylation and ATP production, but also triggers the PCD process (Vianello et al., 2007). It is reported that AgNO3induces
accumulation of ROS and depletion of scavenger enzymes in plants (Vishwakarma et al., 2017). Accordingly, a strong increase of ROS in Ag-treated GSC was observed at 24 h, if compared to control. On the contrary, in AgNO3-treated GSC with lowest concentrations (0.1
and 1 µM), ROS formation tend to decrease after 24 h, in respect to the control 0 µM at 24 h. However, these lower concentrations sig-nificantly reduce ROS formation after 24 h, if compared to control 0 µM at 0 h. This effect could be explained by the activation of several signaling pathways involved in the re-enforcement of antioxidant sys-tem, described in both animals and plants, as reported by Jalmi et al. (2018). We also suggest that the decrease of ROS could depend on the activation of scavenger enzymes or on the synthesis of antioxidant molecules, induced by the transfer of cells from solid media into liq-uid growth medium, which implies a more metabolically active con-dition. Since only 10 µM AgNO3was able to induce a significant ROS
increase, we confirm that ROS and PCD are positively correlated to Ag+ cytotoxicity after 24 h (Figs. 2A and 3 A). On the contrary,
af-ter 6 h incubation with 10 µM AgNO3no difference in ROS
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Fig. 4. Effect of AgNO3and DEVD-CHO on PCD in GSC and DEVD-dependent caspase activity in GSC extracts. A) Caspase activity in GSC grown for 0 or 24 h, in the presence of different
concentrations of AgNO3, respectively. B) Caspase activity in GSC grown for 0, 6 or 24 h in the absence or presence of 10 µM AgNO3. C) PCD in GSC grown for 0 or 24 h, in the presence of
10 µM AgNO3± 10 µM DEVD-CHO. D) Caspase activity in GSC grown for 24 h, in the presence of 10 µM AgNO3± 10 µM DEVD-CHO. Values are means ± S.D. of at least three replicates.
Statistical analysis was the same as in Fig. 2.
level even decreased after 24 h. These data imply the induction of a transitory oxidative stress, probably due to different growth conditions and the presence of light, which the solid cell clumps faced when trans-ferred from solid to liquid media. As demonstrated by Jiao et al. (2014), the supply of antioxidants to tobacco suspension cells reduces the rate of PCD induced by a fungal toxin and allows to restore the redox equilib-rium during stress. This explanation could be applied also to our exper-imental conditions, as confirmed by the increase of antioxidant metabo-lites, such as carotenoids and flavonoids, in GSC samples after 24 h, if compared to what observed at 0 h (Additional file 3, Tables 3 and 4). The correlation between PCD and cell redox status has been long de-bated in the last decade (Huang et al., 2014; Li and Xing, 2011; Van Breusegem and Dat, 2006) and, in most cases, the unbalance among an-tioxidants and ROS becomes a key regulator of PCD (Ge et al., 2016; Han et al., 2012).
In addition to cell shrinkage and DNA laddering, the expression of caspase-like enzymes is considered a suitable marker for detecting cells undergoing both developmental or stress-induced PCD (Ge et al., 2016; Han et al., 2012; Iakimova and Woltering, 2017; Xu and Zhang, 2009). In our system, we also found a strict relationship between PCD occur-rence and DEVD-dependent caspase-3-like activity in GSC treated for 24 h with 10 µM of AgNO3(Fig. 4A). The DEVD-dependent activity was
exerted by a caspase-like enzyme, as confirmed by its sensitivity to the specific inhibitor, when the activity was measured in vitro on cytosolic extracts from 24 h-treated cells (Fig. 4D). In addition, pre-incubation of GSC with the specific DEVD-caspase inhibitor resulted in the decrease of PCD induced by 10 µM AgNO3(Fig. 4C). These findings confirmed
that caspase-3-like activity was associated to Ag+-induced PCD in GSC,
although the involvement of other caspase-like enzymes could not be excluded a priori. The time-course of DEVD-dependent caspase-3-like ac-tivity was similar to the pattern of PCD (cell shrinkage) manifestation and was consistently associated to AgNO3concentration applied to GSC.
This is, to the best of our knowledge, the first report of caspase-3-like involvement in plant PCD induced by Ag+.
The oxidative stress induced by Ag+ may in turn also affect the
UPS, similarly to what described in peanut root tips for Al3+, which
is able to activate caspase-3 like enzymes (Huang et al., 2014). Ac-cordingly, in bean cotyledons the copper-induced lipid peroxidation and hydrogen peroxide accumulation cause UPS impairment and lead to inactivation of proteasome activity, with accumulation of damaged proteins (Karmous et al., 2014). These results are partially in con-flict with those obtained by Polge et al. (2009) in Arabidopsis, where Cd2+induces 20SP synthesis, higher degradation of oxidized proteins
and ultimately high resistance against Cd2+-induced oxidative stress.
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Fig. 5. Effect of AgNO3on the presence of pUCP and PBA1 protein in GSC, detected by immunochemical assay. A) Amount of pUCP in GSC grown for 0 or 24 h, in the presence of different
concentrations of AgNO3, respectively. B) Detection of PBA1 in GSC grown for 0 or 24 h, in the presence of different concentrations of AgNO3, respectively. C) Amount of pUCP in GSC
grown for 6 or 24 h in the absence or presence of 10 µM AgNO3. D) Detection of PBA1 in GSC grown for 6 or 24 h in the absence or presence of 10 µM AgNO3. Values are means ± S.D. of
at least three replicates. Statistical analysis was the same as in Fig. 2.
did not seem to be the primary signal for UPS modulation. In fact, they are not a prerequisite, since the accumulation of pUCP, as a marker of UPS malfunctioning, has been already detected in the presence of 1 µM Ag+after 24 h (Figs. 5A and 6 A), while the same treatment did not
in-duce a significant increase of ROS (Fig. 3A). It is reasonable to hypoth-esize that low Ag+concentration alone directly induced UPS
malfunc-tioning, although PCD manifestation occurred together with oxidative stress and UPS degradation.
The accumulation pattern of pUCP in control and Ag+-treated GSC
showed that a large accumulation of these intermediates took already place after 6 h in both control and treated samples (Figs. 5C and 6C). After 24 h, the control samples reached a quite low level of pUCP, while stressed cells did not. We suggest that during transfer from solid to liquid medium, the cells were subjected to an oxidative and mechan-ical stress in the first hours, which could then be overcome only by untreated cells. On the contrary, in our experimental plan, 1 and 10 µM AgNO3treatments induced an unbalance of the protection system
that prevents UPS degradation and finally resulted in a large pUCP accumulation. The effects observed in GSC were similar to those de-scribed in animal fibroblasts cells, being HMs able to disrupt the com-plex Ub/proteasome, with consequent increase in cellular ubiquitinated protein concentration (Yu et al., 2011). These authors evidenced that the alteration in basal regulation of UPS leads also to unbalance cellu-lar antioxidant systems, by ROS hyper-accumulation and onset of pro-tease enzymatic systems, such as caspase-3-like proteins. Indeed, 10 µM AgNO3induced the degradation of UPS in GSC, as confirmed by
the decrease of PBA1 immunodetection (Figs. 5B and 6B). These data are in agreement with a progressive complete degradation of plant
metabolic machinery due to proteolytic caspase-like activity, similarly to the animal model proposed by Yu et al. (2011). This observation may support, hence, the hypothesis that this mechanism could be well conserved also among different eukaryotic kingdoms. In both models, HMs exert their toxic effects by inducing protein turnover unbalance, together with PCD and proteolytic degradation. In the present work, we found that the decrease of PBA1, induced by high Ag+, affects the
prote-olytic activity of proteasome, as demonstrated by the increase of pUCP. On the other hand, this event did not cause an inhibition or decrement of the whole DEVD-dependent proteolytic activity, in contrast to what found in the characterization of PCD during xylem differentiation (Han et al., 2012). Indeed, it has to be considered that DEVD peptide, used for caspase-3-like activity assay in GSC, is actually a substrate for other iso-forms of caspase-3-like, caspase-7-like and probably caspase-8-like en-zymes (Bieri et al., 2016). These observations in a simplified model sug-gest that proteasome plays an essential function in PCD process also in plants.
5. Conclusions
Our results show that micromolar concentrations of Ag+ induce
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Fig. 6. Western blot analysis of pUCP and PBA1 in protein samples obtained from GSC extracts. A) Western Blot of protein samples obtained from GSC grown for 0 or 24 h, respectively, in the presence of different concentrations of AgNO3, detected by anti-Ub. B) Western Blot of protein samples obtained from GSC grown for 0 or 24 h, respectively, in the presence of
different concentrations of AgNO3, detected by anti-PBA1. C) Western Blot of protein samples obtained from GSC grown for 6 or 24 h, respectively, in the absence or presence of 10 µM
AgNO3, detected by anti-Ub. D) Western Blot of protein samples obtained from GSC grown for 6 or 24 h, respectively, in the absence or presence of 10 µM AgNO3, detected by anti-PBA1.
Protein molecular mass markers are shown on the left.
that also in plants UPS plays a relevant role as a regulator of PCD mani-festation induced by silver contamination.
Authors’ contributions
Antonio Filippi: Methodology, Data curation, Investigation,
Writ-ing-Original draft preparation.
Marco Zancani: Investigation, Writing-Original draft preparation,
Reviewing and Editing.
Elisa Petrussa: Conceptualization, Supervision, Methodology, Data
curation, Investigation, Validation, Writing-Original draft preparation, Reviewing and Editing. Enrico Braidot: Conceptualization, Supervision, Formal Analysis, Visualization, Writing-Original draft preparation, Re-viewing and Editing, Project Administration.
All authors have read and approved the final version of the manu-script.
Funding
This research did not receive any specific grant from funding agen-cies in the public, commercial, or not-for-profit sectors.
Declarations of interest
None
Acknowledgements
We thank Dr. Vladimír Repka for providing the grapevine cell cul-tures, and Dr. Alberto Bertolini and Dr. Gabriele Bradamante for their contribution to some experiments.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jplph.2018.12.003.
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